Ischemic coronary artery disease is one of the leading causes of morbidity and mortality in the United States. Current therapeutic approaches include risk factor modification, reduction of myocardial oxygen demand, and localized restoration of flow to the coronary arterial system by means of angioplasty, stents, or coronary bypass. These treatment options are often insufficient and a significant number of patients are not even candidates for the surgical options. Some attempts have been made to improve coronary flow blood supply to ischemic myocardium by grafting or by measures to enlarge existing blood vessels.
Therapeutic angiogenesis is a process which can be used to develop collateral blood vessels to the myocardium. The term “angiogenesis” refers to a process of tissue vascularization that involves the development of new vessels. Angiogenesis is a complex process involving the breakdown of extracellular matrix, with proliferation and migration of endothelial and smooth muscle cells ultimately resulting in the formation and organization of new blood vessels (Folkman, J., and Klagsbrun, M., Science 235:442-7, 1987). Angiogenesis typically occurs via one of three mechanisms: (1) neovascularization, where endothelial cells migrate out of pre-existing vessels beginning the formation of the new vessels; (2) vasculogenesis, where the vessels arise from precursor cells de novo; or (3) vascular expansion, where existing small vessels enlarge in diameter to form larger vessels (Blood, C. H., and Zetter, B. R., Biochem. Biophys. Acta. 1032:89-118, 1990). Angiogenesis is an important process in normal processes of neonatal growth and in the female reproductive system during the corpus luteum growth cycle (see Moses, M. A., et al., Science 248: 1408-10, 1990). Under normal conditions, all processes involving the new formation or the remodeling of existing or new blood vessels is a self-limiting process, and the expansion of the specific cell types is controlled and concerted. Angiogenesis is also involved in wound healing and in the pathogenesis of a large number of clinical diseases including tissue inflammation, arthritis, asthma, tumor growth, diabetic retinopathy, and other conditions. Clinical manifestations associated with angiogenesis are referred to as angiogenic diseases (Folkman and Klagsbrun, 1987, supra).
The term “growth factors” originally referred to substances that promote cell growth. It is used to indicate molecules that function as growth simulators (mitogens) but also as growth inhibitors (sometimes referred to as negative growth factors). Growth factors are also known to stimulate cell migration (e.g., mitogenic cytokines ), function as chemotactic agents, inhibit cell migration or invasion of tumor cells, modulate differentiated functions of cells, be involved in apoptosis and promote survival of cells. Such factors can be secreted as diffusible factors and can also exist in membrane-anchored forms. They can, therefore, act in an autocrine, paracrine, juxtacrine or retrocrine manner. A cytokine is one type of growth factor.
The term “cytokine” is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Cytokines comprise interleukins, initially thought to be produced exclusively by leukocytes; lymphokines, initially thought to be produced exclusively by lymphocytes; monokines, initially thought to be produced exclusively by monocytes; interferons, initially thought to be involved in antiviral responses; colony stimulating factors, initially thought to support the growth of cells in semisolid media; and chemokines, thought to be involved in chemotaxis, and a variety of other proteins. In vivo, the expression of most cytokines is strictly regulated; these factors are usually produced only by activated cells in response to an induction signal.
In general, cytokines act on a wider spectrum of target cells than hormones. Perhaps the major feature distinguishing cytokines from mediators regarded generally as hormones is the fact that, unlike hormones, cytokines are not produced by specialized cells which are organized in specialized glands; there is not a single organ source for these mediators. The fact that cytokines are secreted proteins also means that the sites of their expression does not necessarily predict the sites at which they exert their biological function. Some cytokines have been found, upon determination of their primary structures, to be identical with classical enzymes (for example: adult T-cell leukemia-derived factor (ADF), nm23, platelet-derived endothelial cell growth factor (PD-ECGF, or neuroleukin). Cytokines normally do not possess enzymatic activities although there is a growing list of exceptions. The biological activities of cytokines can be measured by a variety of bioassays employing, among other things, factor-dependent cell lines, or cytokines can be measured by other assays using, for example, antibodies. Message amplification phenotyping employs modem techniques of molecular biology and detects the presence of cytokine-specific mRNAs.
Many experiments have suggested that tissues can produce angiogenic factors which promote angiogenesis under conditions of poor blood supply during both normal and pathological conditions. Several angiogenic factors have been demonstrated in vivo to promote angiogenesis in the ischemic myocardium, including basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), amongst cytokines and other molecules (Lopez and Simons, Drug Delivery 3:143, 1996). These factors and compounds differ in cell specificity and in the mechanisms by which they induce the growth of new blood vessels. For example, they may induce the migration and proliferation of endothelial cells or stimulate the production collagenase (see Klagsbrun, M., and D'Amore, P. A., Ann. Rev. Physiol. 53:217-39, 1991). There are a number of bioassays which allow direct determination of angiogenic activities (Wilting, J., et al., Anat. Embrol. (Berl) 183:259-71, 1991).
Vascular endothelial growth factor (VEGF) is a homodimeric, heavily glycosylated protein of 46-48 kDa (24 kDa subunits) (for review see Ferrara, N., et al., J. Cell Bio. 47:211, 1991; Ferrara, N., et al., Endocrin. Rev. 13:18-32, 1991). Glycosylation is not required, however, for biological activity. The subunits are linked by disulphide bonds. The human factor occurs in several molecular variants of 121, 165, 189, and 206 amino acids, arising by alternative splicing of the mRNA. Several other variants of VEGF have been described, including VEGF-B, VEGF-C, and VEGF-D. The 189 amino acid variant of VEGF (VEGF-189) is identical with vascular permeability factor (VPF). VEGF-121 and VEGF-165 are soluble secreted forms of the factor while VEGF-189 and VEGF-206 are mostly bound to heparin-containing proteoglycans in the cell surface or in the basement membrane. Rat and bovine VEGF are one amino acid shorter than the human factor, and the bovine and human sequences show a homology of 95%. The positions of all eight cysteine residues are conserved in VEGF and PDGF. A high-affinity glycoprotein receptor for VEGF of 170-235 kDa is expressed on vascular endothelial cells. VEGF significantly influences vascular permeability and is a strong angiogenic protein in several bioassays, and has been shown to be a highly specific mitogen for vascular endothelial cells. In vitro, the two shorter forms of VEGF stimulate the proliferation of macrovascular endothelial cells, but does not appear to enhance the proliferation of other cell types.
Perivascular delivery of bFGF has been shown to improve collateral circulation and myocardial function in chronic myocardial ischemia (Harada et al., J. Clin. Invest. 94:623-30, 1994). Both bFGF and VEGF have been shown to enhance collateral blood flow during perivascular delivery by myocardial perfusion by heparin alginate microspheres and implantable osmotic pumps (Lopez and Simmons, 1996, supra). However, the systemic delivery of growth factors by means such as intravenous infusion has a number of limitations. A number of adverse effects have been described with the systemic administration of angiogenic growth factors, including renal and hematopoietic end-organ damage such as membranous nephropathy and bone marrow suppression as well as hemodynamic effects (Lopez and Simmons, 1996, supra). In addition, concerns have been raised as to the potential of systemic delivery of these agents to stimulate dormant neoplasias. The cost of the systemic delivery of recombinant growth factor proteins is also thought to be prohibitive.